1.1 Field of the Invention
The present invention relates generally to the field of oncology. More particularly, certain embodiments concern methods for making and using lipid-carrier protein conjugate compositions such as phosphatidylserine (PS)-conjugates for generating lipid-specific immune responses in an animal. Also disclosed are methods for making PS antigen and antibody compositions and their use in a variety of therapeutic applications, including the formulation of pharmaceutical compositions for the prevention and treatment of cancers.
1.2 Description of the Related Art
The results of many studies have led to the concept that membrane phospholipid asymmetry is ubiquitous. The outer leaflet of eukaryotic plasma membranes contains most of the cholinephospholipids, whereas the aminophospholipids are mainly present in the cell's inner leaflet (Devaux, 1991; Schroit and Zwaal, 1991). While asymmetry seems to be the rule for normal cells, loss of membrane lipid sidedness, in particular the emergence of phosphatidylserine (PS) at the cell surface, results in the expression of altered surface properties that modulates cell function and influences the cells interaction with its environment (Zwaal and Schroit, 1997). For example, the exposure of PS promotes coagulation and thrombosis by platelets (Bevers et al., 1983; Rosing et al., 1985; Thiagarajan and Tait, 1990) and the recognition of apoptotic (Fadok et al, 1992; Bennett et al, 1995; Sambrano and Steinberg, 1995; Verhoven et al, 1995) and aged (Herrmann and Devaux, 1990; Geldwerth et al., 1993; Connor et al 1994) cells by the reticuloendothelial system.
To characterize these and other PS-related processes, new tools are required to determine physiologically-dependent alterations in the distribution of PS in cell membranes. Although the application of classical biochemical methodologies (Gordesky et al., 1975; Schick et al., 1976; Etemadi, 1980; Bevers et al., 1982) has yielded important information on PS asymmetry, most of these methods are invasive and destructive. Recently developed methods, such as the PS-dependent prothrombinase assay (Bevers et al., 1983; Rosing et al., 1980; Van Dieijen et al., 1981) and labeled annexin V binding (Thiagarajan and Tait, 1990; Tait and Gibson, 1994; Vermes et al., 1995; Kuypers et al., 1996), are non-invasive and have provided the means to assess the presence and topology of PS in the outer leaflet of viable cells. These methods, however, require the inclusion of various plasma cofactors and/or divalent cations which might influence the lateral distribution of lipids in the plane of the membrane.
While antibodies against different membrane components has become an indispensable aid in the study of membrane structure and function, little attention has been given to the application of lipid-specific antibodies for studying lipid-dependent processes. Because of the inherent difficulty of producing antibodies against small highly conserved lipids, the development of lipid antibodies has progressed slowly. Nonetheless, several laboratories have produced antibodies against certain phospholipid species by immunization with liposomes (Maneta-Peyret et al., 1988; Maneta-Peyret et al., 1989; Banedi and Alving, 1990) or by adsorption of monomeric phospholipids to proteins (Maneta-Peyret et al., 1989; Tamamura et al., 1971), bacteria (Umeda et al., 1989) and acrylamide (Maneta-Peyret et al., 1988; Maneta-Peyret et al., 1989). Antibodies produced by these methods, however, may cross-react with different lipids (Banedi and Alving, 1990; Umeda et al., 1989) and other phosphate-containing moieties (Alving, 1986).
1.3 Deficiencies in the Prior Art
While some methods have been developed in these areas, what is lacking in the prior art are effective methodologies for generating immune responses that are useful in various treatment regimens, including those specific for oncology.
Several reports on the production of PS antibodies have been published. These include unrelated methods and approaches using PS-containing liposomes (Banerji and Alving, 1990), PS-coated Salmonella (Umeda et al., 1989) and acrylamide-entrapped PS (Maneta-Peyret et al., 1988). There is one report concerning carrier (KLH)-coupled PS (Bate et al., 1993). However, the chemistry employed to make the conjugate couples lipid to the protein carrier via the lipids primary amine thereby destroying antigenic specificity. Immunization with this conjugate produced antibody activity which inhibited the production of tumor necrosis factor by malaria-infected erythrocytes. Whether the antibodies had any lipid specificity was not investigated. This is unlikely, however, considering that the conjugation chemistry destroyed the key primary amine determinant of phosphatidylserine. Thus, there exists an immediate need for an effective method of producing highly-specific anti-PS antibodies and cell-mediated PS responses for use in the diagnosis and treatment of various cancers and related conditions.